The boy who put his finger in a dam eventually got wet. Like dripping faucets, most inducible expression systems leak gene expression in the off state. Now Deans et al. report a transgene expression system that plugs leaky expression in a recent article in Cell.
Researchers have designed several inducible expression systems. The E. coli tetracycline repressor (TetR) suppresses expression of genes linked to the tetracycline operator (tetO). Tetracycline-inducible gene expression systems exploit this interaction. However, genetic repressors, including TetR and LacI, do not completely block gene expression. RNA interference (RNAi) mediated by small interfering (si) or short-hairpin (sh) RNA blocks gene expression, but is difficult to design and can have unintended off-target effects in the genome.
The authors designed a gene expression system, called Lac-Tet-RNAi (LTRi) that suppresses transgene expression in the off state with repressor proteins and RNAi. They engineered a lac operator (lacO) site upstream and a synthetic shRNA target sequence downstream of the transgene of interest. The single-plasmid expression network also included shRNA under the control of tetO, TetR under the control of lacO and LacI. In the default (off) state, Lacl suppresses expression of TetR and the transgene transcript. Any leaky transgene expression would be blocked by shRNA, which has few unintended effects because of its synthetic target. The lactose metabolite isopropyl-
-thiogalactopyranoside (IPTG) alters the conformation of LacI, preventing its action at lacO. By inhibiting LacI, IPTG turns on transgene expression and induces TetR, which suppresses shRNA production.
Relative to cells transfected with an enhanced green fluorescent protein (EGFP) transgene alone, cells transfected with the shRNA or LacI module respectively showed 80% and 85% reductions in fluorescence, suggesting that both shRNA and Lac repression allow leaky expression. In contrast, cells transfected with the full LTRi gene network showed more than 99% reduction in EGFP expression relative to EGFP-transfected control. Three days of IPTG treatment induced peak EGFP induction, which was 85 times baseline expression level. Within three days of IPTG removal, EGFP expression returned to baseline. The level of transgene induction was dependent on the amount of IPTG used, rather than expression history. Low levels of IPTG (250 pM) induced low EGFP expression, even if cells had previously received high levels of IPTG (1 mM) and shown high EGFP expression.
The authors used LTRi to induce toxin expression. The 
chain of diptheria toxin (DTA) is so toxic that even one molecule can kill a cell. The authors inserted a DTA transgene into LTRi. Despite DTA's toxicity, cells expressing the gene network survived for at least four weeks. However, IPTG treatment induced cell death. These data suggest that LTRi blocks leaky expression completely.
According to the authors, LTRi-induced expression of toxic proteins may be targeted, allowing fine-tuned control of brain lesions.